WO2012145382A2 - Mesure de pka pour caractérisation d'un cancer - Google Patents

Mesure de pka pour caractérisation d'un cancer Download PDF

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WO2012145382A2
WO2012145382A2 PCT/US2012/034049 US2012034049W WO2012145382A2 WO 2012145382 A2 WO2012145382 A2 WO 2012145382A2 US 2012034049 W US2012034049 W US 2012034049W WO 2012145382 A2 WO2012145382 A2 WO 2012145382A2
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pka
activity
mixture
carcinoma
sample
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WO2012145382A3 (fr
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Robert Puskas
Douglas Held
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Traxxsson LLC
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Traxxsson LLC
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Publication of WO2012145382A3 publication Critical patent/WO2012145382A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/57585—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds identifiable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57557—Immunoassay; Biospecific binding assay; Materials therefor for cancer of other specific parts of the body, e.g. brain
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention generally relates to medical diagnostics and to methods, kits and assays for the determination of certain protein biomarkers and the characterization of cancer.
  • PKA cAMP-dependent protein kinase
  • R regulatory subunit
  • C active catalytic kinase subunit
  • Most of the effects of cAMP in eukaryotic systems are the result of phosphorylation of proteins at serine or threonine residues by PKA.
  • PKA is localized subcellularly by binding to multidomain scaffolding proteins known as AKAPs which bind to the R subunits of the holoenzyme [1 ]. More than 50 AKAPs are known which localize PKA in various cell types.
  • the PKA-specific inhibitor (PKI) acts by binding with high affinity to the substrate binding site of the free active catalytic subunit [2].
  • the PSA (prostate-specific antigen) blood test for prostate cancer is of questionable value.
  • the American Cancer Society no longer recommends that men routinely have PSA tests as part of their routine physical examinations [7].
  • the PSA test fails to detect 3 out of 4 cases [8].
  • the PSA test comes back positive, 3 out 4 times it is a false positive - the patient does not have cancer [9].
  • patients who have a positive PSA test typically will have a group of 12 or more biopsy samples taken from their prostate to verify if cancer is present.
  • mammograms are purported to detect 85-90% of breast cancers when they are present, the detected tumors on average are 1 1 /2 inches in diameter when diagnosed. As for all cancers, earlier detection leads to better patient outcomes. What makes breast cancer screening costly is that an estimated 95% of the positive mammograms are false positives - the patient does not have cancer [12].
  • a positive mammogram frequently leads to a breast biopsy. A typical needle biopsy costs about $1 ,500; an invasive surgical biopsy (about 1/3 of all breast biopsies) costs about $5,000. This brings the national cost for the estimated 2 million unnecessary breast biopsies to more than $5.1 billion annually. Improved patient outcome could be accomplished by substituting a cancer confirmatory test for a biopsy, for a savings in health care costs of almost $5 billion annually
  • Cancer monitoring tests Beyond screening tests there are additional blood tests that are used to monitor cancer patients once cancer has been diagnosed. Many of these tests are not specific for cancer or specific for a particular type of cancer, rendering them of limited value as cancer screening tests. However, they can be an effective means for monitoring cancer treatment and testing for disease recurrence.
  • extracellular PKA extracellular PKA
  • xPKA extracellular PKA
  • extracellular PKA derived from a bodily fluid of a subject afflicted with a carcinoma and extracellular PKA derived from a bodily fluid of a subject not afflicted with a carcinoma can exhibit significantly different activities for the phosphorylation of a PKA substrate and, thus, can be used to characterize carcinoma in a subject.
  • one aspect of the present invention is directed to a method for characterizing a carcinoma in a subject.
  • the method comprises assaying a sample of a bodily fluid derived from the subject for extracellular PKA activity, the assay comprising preparing a reaction mixture comprising the previously unfrozen sample, a PKA peptide substrate, a phosphorylation agent, incubating the prepared mixture, and detecting phosphorylated substrate formed in the incubated mixture.
  • the amount of phosphorylated substrate formed in the assay is compared to a reference value, the reference value being the amount of phosphorylated substrate formed in a mixture under equivalent conditions for a sample of bodily fluid derived from a population of subjects not afflicted with cancer.
  • the present invention is further directed to a kit for determining the amount of extracellular PKA activity in a sample.
  • the kit comprises a PKA substrate, a phosphorylation agent, and control PKA of known activity.
  • the kit further comprises an oxidizing agent.
  • the kit further comprises a phosphatase inhibitor.
  • Fig. 1 is graph depicting the effect of oxidation reduction potential (mV) upon the ratio of apparent xPKA activity (cancer subjects/normal subjects) as more fully described in Example 1 .
  • Fig. 2 is graph depicting the effect of oxidation reduction potential (mV) upon the ratio of apparent xPKA activity (cancer subjects/normal subjects) as more fully described in Example 2.
  • Fig. 3 is graph depicting xPKA activity levels for samples with NaF (phosphatase inhibitor) in the reaction mixture as more fully described in Example 3.
  • Fig. 4 is graph depicting oxidation reduction potential and ratios of Cancer/Normal xPKA activity with NaF (phosphatase inhibitor) in the reaction mixture as more fully described in Example 3.
  • Fig. 5 is a table presenting apparent xPKA activities in samples from prostate and colon cancer patients relative to those from individuals apparently without cancer with various concentrations of oxidant or reductant used in the sample preparation buffer as more fully described in Example 1 .
  • extracellular PKA or "xPKA” means cAMP-dependent protein kinase A found in bodily fluids outside of bodily cells.
  • normal subject or "normal individual” means a subject or individual not known to be afflicted with, or suspected of being afflicted with, a carcinoma.
  • ORP means oxidation-reduction potential
  • extracellular PKA may be used to characterize carcinoma in a subject. More specifically, it has been determined that extracellular PKA derived from persons unafflicted with carcinoma and extracellular PKA derived from persons afflicted with carcinoma can be differentiated, depending upon the reaction conditions. Under certain reaction conditions, extracellular PKA derived from persons unafflicted with carcinoma has a greater activity for the phosphorylation of a PKA substrate than does extracellular PKA derived from persons afflicted with carcinoma. Under certain other reaction conditions, extracellular PKA derived from persons unafflicted with carcinoma has less activity for the phosphorylation of a PKA substrate than does extracellular PKA derived from persons afflicted with carcinoma. Under yet other reaction conditions, extracellular PKA derived from persons unafflicted with carcinoma and extracellular PKA derived from persons afflicted with carcinoma have approximately equivalent activities for the phosphorylation of a PKA substrate.
  • the results of an assay for an individual subject under a set of reaction conditions may be compared to a reference value for that set of reaction conditions to characterize carcinoma in that subject. For example, if the assay for the individual subject is carried out under reaction conditions at which the activity of extracellular PKA derived from subjects afflicted with carcinoma for the phosphorylation of a PKA substrate is significantly greater than the activity of extracellular PKA derived from subjects unafflicted with carcinoma for the phosphorylation of a PKA substrate (i.e., normal subjects), and the activity of the individual subject's xPKA for the phosphorylation of PKA substrate is significantly greater than the activity that is characteristic of normal subjects, a diagnosis, prognosis, etc., may be determined.
  • the assay for the individual subject is carried out under reaction conditions at which the activity of extracellular PKA derived from subjects afflicted with carcinoma for the phosphorylation of a PKA substrate is significantly less than the activity of extracellular PKA derived from normal subjects for the phosphorylation of a PKA substrate, and the activity of the individual subject's xPKA for the phosphorylation of PKA substrate is significantly less than the activity that is characteristic of subjects unafflicted with carcinoma, a diagnosis, prognosis, etc., may be determined.
  • the relative activities of extracellular PKA derived from subjects afflicted with carcinoma and normal individuals for the phosphorylation of a PKA substrate depends, at least in part, upon the oxidation state of the PKA in the assay.
  • the activity of extracellular PKA from normal individuals appears to be significantly influenced by the redox environment in which it is found.
  • Extracellular PKA from normal individuals appears to have lower activity when the redox environment is oxidizing and higher activity when the environment is highly reducing. Consequently, the apparent activity of extracellular PKA in a fluid sample derived from normal individuals can be increased by treating the sample with a reducing agent to form a mixture that has an ORP value that is more reducing, or decreased by treating the sample with an oxidizing agent.
  • the activity of extracellular PKA derived from individuals afflicted with a carcinoma is relatively insensitive to oxidation state. That is, the activity is relatively constant irrespective of whether it is treated with an oxidizing agent or a reducing agent.
  • apparent PKA activity is assayed under moderately reducing conditions, i.e., the range of conditions at which the apparent xPKA activity in samples derived from cancer patients is greater than the apparent xPKA activity that is characteristic of normal patients.
  • Moderately reducing conditions may be established, for example, by forming a mixture comprising the sample and a reducing agent wherein the mixture has an ORP value in the range of about -1 10 mV to about -20 mV.
  • the mixture containing the sample has an ORP value in the range of about -100 mV to about -90 mV.
  • the mixture containing the sample has an ORP value in the range of about -20 mV to about -30 mV.
  • apparent PKA activity is assayed under oxidizing conditions, or moderately or highly reducing conditions, i.e., the range of conditions at which the apparent xPKA activity in samples derived from cancer patients is less than the apparent xPKA activity that is characteristic of normal patients.
  • Highly reducing conditions may be established, for example, by forming a mixture comprising the sample and a reducing agent wherein the mixture has an ORP value that is less than about -1 10 mV (that is, conditions that are more reducing than about -1 10 mV).
  • the mixture containing the sample has an ORP value of less than about -120 mV.
  • the mixture containing the sample has an ORP value of less than about -145 mV.
  • moderately reducing or oxidizing conditions may be established, for example, by forming a mixture comprising the sample and an oxidizing agent or a reducing agent wherein the mixture has an ORP value of greater than -20 mV (that is, conditions that are more oxidizing than -20 mV).
  • the mixture containing the sample has an ORP value of at least about -15 mV.
  • the mixture containing the sample has an ORP value of at least about 1 mV).
  • the mixture containing the sample has an ORP value of at least about 60 mV).
  • the mixture containing the sample has an ORP value of at least about 128 mV.
  • the sample may be incubated in a mixture having a desired, or at least known redox environment, for a period of time, before the assay is initiated.
  • the incubation time will be at least about 1 minute before the PKA activity assay is initiated.
  • greater incubation times will be employed.
  • the incubation time will be at least about 5 minutes.
  • the incubation time will be at least 10 minutes.
  • the incubation time will be at least 30 minutes.
  • the incubation time will be at least about 1 hour.
  • the incubation temperature may be in the range of 20 to 37 °C, with about 25 °C being preferred in certain embodiments. This may be accomplished, for example, in an incubation mixture formed prior to the combination of the sample with the PKA substrate.
  • the sample is not incubated with an oxidizing agent or a reducing agent for a period of time before the PKA activity assay is initiated. Rather, a reaction mixture for determining PKA activity is prepared directly from the sample by combining the sample with a PKA peptide substrate, a phosphorylation agent, and optionally a reducing agent or oxidizing agent, the prepared mixture is incubated, and phosphorylated substrate formed in the incubated mixture is detected.
  • the assay be carried out under reaction conditions at which a ratio of the activities for a statistically significant population of normal subjects to a statistically significant population of subjects afflicted with carcinoma be at least 0.05:1 and less than 0.8:1 . In certain embodiments, it is preferred that the ratio of the activities for a statistically significant population of normal subjects to a statistically significant population of subjects afflicted with carcinoma be at least 0.075:1 and less than 0.6:1 . In certain embodiments, it is preferred that the ratio of the activities for a statistically significant population of normal subjects to a statistically significant population of subjects afflicted with carcinoma be at least 0.1 and less than 0.4:1 .
  • the reaction conditions for an assay be those at which the activities are significantly different. That is, it is generally preferred that the assay be carried out under reaction conditions at which a ratio of the activities for a statistically significant population of normal subjects to a statistically significant population of subjects afflicted with carcinoma be at least 1 .2:1 or less than 0.8:1 . In certain embodiments, it is preferred that the ratio of the activities for a statistically significant population of normal subjects to a statistically significant population of subjects afflicted with carcinoma be at least 2:1 or less than 0.5:1 . In certain embodiments, it is preferred that the ratio of the activities for a statistically significant population of normal subjects to a statistically significant population of subjects afflicted with carcinoma be at least 3:1 or less than 0.2:1 .
  • PKA has two exposed cysteines Cys 199 and Cys 343 . It has been determined that sulfhydryl modification of Cys 343 has minimal impact on enzyme activity. However, sulfhydryl modification of Cys 199 predisposes the enzyme to dephosphorylation and inactivation [1 ]. Humphries et al demonstrated that apparent PKA activity in cell lysates is regulated by an interplay between oxidation of PKA and oxidation of phosphatases. [2] Because these enzymes differentially respond to oxidation, they react differentially to the redox state of the sample, and the overall apparent activity of PKA varies in a complex manner in response to the redox state of a sample.
  • the results of the assay may be used to characterize a carcinoma in a subject. That is, the assay may be used to detect or diagnose cancer. In certain embodiments, it may also be used for the determination of a prognosis, determination of drug efficacy, monitoring the status of said subject's response or resistance to a treatment or selection of a treatment for said carcinoma.
  • the carcinoma may be, for example, a carcinoma selected from the group consisting of lung, colon, pancreatic, ovarian, bladder, liver, and prostate cancer.
  • the methods of the present invention may advantageously be used to characterize a carcinoma or otherwise assess xPKA activity in a variety of subjects.
  • the subject may be, for example, a mammal such as bovine, avian, canine, equine, feline, ovine, porcine, or primate (including humans and non-human primates).
  • a subject may also include mammals of importance due to being endangered, or economic
  • the subject is a human subject.
  • the subject is bovine, avian, canine, equine, feline, ovine, porcine, or non-human primate.
  • the subject may have a pre-existing disease or condition, such as cancer. Alternatively, the subject may not have any known pre-existing condition. The subject may also be non-responsive to an existing or past treatment, such as a treatment for cancer.
  • reference values shall be for members of a given species.
  • xPKA values for human subjects shall only be compared to xPKA values for a statistically significant population of human subjects under equivalent assay conditions.
  • xPKA values for non-human subjects shall only be compared to xPKA values for a statistically significant population of non-human subjects of the same species under equivalent assay conditions.
  • assays for determination of the activity of extracellular PKA may be carried out by preparing a reaction mixture comprising the sample, a
  • the fluid sample may be derived from any bodily fluid of a subject or subjects. In one embodiment, the sample is previously unfrozen.
  • Exemplary bodily fluids include peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates or other lavage fluids.
  • CSF cerebrospinal fluid
  • saliva bone marrow
  • synovial fluid aqueous humor
  • amniotic fluid cerumen
  • breast milk
  • Additional exemplary bodily fluids include the blastocyl cavity, umbilical cord blood, or maternal circulation which may be of fetal or maternal origin.
  • the fluid sample is derived from a bodily fluid selected from among whole blood, sputum, serum, plasma, urine, cerebrospinal fluid, nipple aspirate, saliva, fine needle aspirate, and combinations thereof.
  • a bodily fluid selected from among whole blood, sputum, serum, plasma, urine, cerebrospinal fluid, nipple aspirate, saliva, fine needle aspirate, and combinations thereof.
  • the fluid sample is derived from a bodily fluid selected from among whole blood, serum, plasma, urine, nipple aspirate, saliva, and combinations thereof.
  • the fluid sample is derived from blood plasma or serum.
  • the sample is treated with a reducing agent or an oxidizing agent.
  • This treatment step may be carried out before the sample is combined with the other components of the reaction mixture, or along with the other components of the reaction mixture.
  • Reducing agents such as 2-mercaptoethanol, Syringaldazine, sodium hydrosulfite, dithiothreitol, dithioerythreitol,and tris(2-carboxyethyl)phosphine
  • TCEP hydrochloride
  • the reducing agents may preferably be used be at concentrations between 50 ⁇ and 500 mM; in one embodiment, the reducing agents may preferably be used be at concentrations between 50 ⁇ and 100 mM.
  • the reducing agents may be mixed with the sample prior to addition of the sample to the assay, or the reducing agents may be incorporated into the assay mixture.
  • the sample, separately or in the reaction mixture, may be incubated with the reducing agents preferably between 1 minute and 60 minutes.
  • Oxidizing agents such as diamide, or hydrogen peroxide may be used as oxidizing agents to affect the redox state of the PKA reaction.
  • the oxidizing agents may preferably be used be at concentrations between 5 uM and 100 mM.
  • the reducing agents may be mixed with the sample prior to addition of the sample to the assay, or the reducing agents may be incorporated into the assay mixture.
  • the sample, separately or in the reaction mixture, may be incubated with the reducing agents preferably between 1 minute and 60 minutes
  • the phosphorylation agent will typically be ATP although other phosphorylation agents may be employed in certain embodiments.
  • the PKA substrate may be any peptide substrate for PKA.
  • Exemplary PKA substrates include histone lla.
  • the PKA substrate is Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly).
  • a specific inhibitor of PKA may be useful to discriminate PKA activity from other related kinase activity.
  • PKA-specific inhibitor which may be used for this purpose is PKI peptide (lle-Ala-Ala-Gly-Arg-Thr-Gly-Arg-Arg-Gln-Ala-lle-His-Asp-lle- Leu-Val-Ala-Ala-OH).
  • Related peptides and shorter peptides derived from the PKI sequence also may be used as PKA-specific inhibitors.
  • the phosphorylated substrate may be detected using a variety of systems.
  • a probe having affinity for the phosphorylated substrate is conjugated to a "functional group" which is directly or indirectly detectable.
  • the probe may be, for example, an antiphosphoserine antibody.
  • the functional group may be a moiety which is measurable by direct or indirect means (e.g., a radiolabel, a
  • the functional group may be a moiety that is indirectly detectable such as an enzyme (e.g., horse radish peroxidase, alkaline phosphatase etc.), biotin, or a hapten such as digoxigenin.
  • an enzyme e.g., horse radish peroxidase, alkaline phosphatase etc.
  • biotin e.g., biotin
  • a hapten such as digoxigenin.
  • an antibody probe is conjugated to a functional group such as a radiolabel, fluorophore, chromophore, chemiluminescent moiety, or enzyme, to facilitate detection.
  • the probe is conjugated to one member of an affinity pair, e.g., biotin, and a detectable label is conjugated to the second member of the affinity pair, e.g., avidin or streptavidin.
  • radiolabels include 3 H, 125 l, 35 S, 14 C, 32 P, and 33 P.
  • Exemplary chromophore/luminophores include any organic or inorganic dyes, fluorophores, phosphophores, light absorbing nanoparticles (e.g., Au, Ag, Pt, Pd), combinations thereof, or the metalated complexes thereof.
  • Exemplary organic dyes are selected from the group consisting of coumarins, pyrene, cyanines, benzenes, N-methylcarbazole, erythrosin B, N-acetyl-L- tryptophanamide, 2,5-diphenyloxazole, rubrene, and N-(3-sulfopropyl)achdinium.
  • preferred coumarins include 7-aminocoumarin, 7-dialkylamino coumarin, and coumarin 153.
  • exemplary benzenes include 1 ,4-bis(5-phenyloxazol-2- yl)benzene and 1 ,4-diphenylbenzene.
  • exemplary cyanines include oxacyanines, thiacyanines, indocyanins, merocyanines, and carbocyanines.
  • exemplary cyanines include ECL Plus, ECF, C3-Oxacyanine, C3-Thiacyanine Dye (EtOH), C3- Thiacyanine Dye (PrOH), C5-lndocyanine, C5-Oxacyanine, C5-Thiacyanine, C7-
  • Exemplary inorganic dyes include metalated and non-metalated porphyrins, phthalocyanines, chlorins (e.g., chlorophyll A and B), and metalated chromophores.
  • Exemplary porphyrins include porphyrins selected from the group consisting of tetra carboxy-phenyl-porphyrin (TCPP) and Zn-TCPP.
  • Exemplary metalated chromophores include ruthenium polypyridyl complexes, osmium polypyridyl complexes, rhodium polypyridyl complexes, 3-(1 -methylbenzoimidazol-2-yl)-7- (diethylamino)-coumarin complexes of iridium(l l l), and 3-(benzothiazol-2-yl)-7- (diethylamino)-coumarin complexes with iridium(lll).
  • Exemplary fluorophores and phosphophores include phosphorescent dyes, fluoresceines, rhodamines (e.g., rhodamine B, rhodamine 6G), and anthracenes ⁇ e.g., 9-cyanoanthracene, 9,10-diphenylanthracene, 1 -Chloro-9,10- bis(phenylethynyl)anthracene).
  • Serum samples from patients with cancer and individuals apparently without cancer were obtained from ProMedDx, LLC and from ProteoGenex.
  • blood samples from prostate cancer patients and normal controls presumably without cancer were assayed for apparent xPKA activity.
  • extracellular PKA in samples was mixed with a defined peptide used as a substrate. The substrate peptide was bound to the wells of the microtiter assay plate.
  • Phosphorylation of the peptide was detected using biotinylated phosphoserine antibody, which was in turn was detected in an ELISA format using peroxidase-conjugated to streptavidin. Detection of the bound peroxidase was established using a color- producing peroxidase substrate included in the assay kit. Bovine PKA catalytic unit was used at varying concentrations to develop a standard activity curve. The detail of the assay protocol is described below.
  • PKA diluent 25 mM KH 2 P0 4 , 5 mM EDTA, 150 mM NaCI, 50% (w/v) glycerol, 1 mg/ml BSA, and various concentrations of reductant or oxidant (2-mercaptoethanol, dithiothreitol, dithioerythritol, or diamide) as indicated directly in the data figures by concentration or by oxidation-reduction potential, pH 6.5
  • aa Perform a least squares linear regression on the data or use a third order polynomial curve (cubic spline) on the data to determine a standard curve bb. Determine kinase concentration in the samples from interpolation of the values on the standard curve
  • ORP oxidation-reduction potential
  • FIG. 5 shows the relative apparent xPKA activities in samples from prostate and colon cancer patients relative to those from individuals apparently without cancer with various concentrations of oxidant or reductant used in the sample preparation buffer.
  • the same pattern of xPKA activity is observed with both the prostate and the colon cancer patient samples.
  • the relative PKA activities of cancer patients is higher, lower, or the same as that of individuals apparently without cancer, depending upon the concentration of oxidant or reductant used in the sample preparation buffer.
  • xPKA activities were determined using the procedure described in Example 1 with the exception that oxidant addition, reductant addition, or no addition was made to the reaction buffer. Samples were not preincubated in sample buffer, but were incubated for 5 minutes at 25 C in reaction buffer with shaking at 750 rpm prior to adding the reaction mixtures to the assay plate wells.
  • ORP oxidation-reduction potential
  • AMERICAN CANCER SOCIETY CANCER FACTS & FIGURES 2008. 2008, AMERICAN CANCER SOCIETY: ATLANTA.

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Abstract

La présente invention concerne un procédé pour caractériser un carcinome chez un sujet. Le procédé comprend l'analyse d'un échantillon de fluide corporel dérivé du sujet pour l'activité PKA extracellulaire et la comparaison de l'activité à une valeur de référence. Dans l'analyse, un mélange de réaction est préparé comprenant l'échantillon précédemment décongelé, un substrat peptidique de PKA, un agent de phosphorylation, le mélange préparé est incubé, et le substrat phosphorylé formé dans le mélange incubé est détecté. La valeur de référence est la quantité de substrat phosphorylé formé dans un mélange dans des conditions redox équivalentes pour un échantillon de fluide corporel dérivé d'une population de sujets normaux de la même espèce.
PCT/US2012/034049 2011-04-20 2012-04-18 Mesure de pka pour caractérisation d'un cancer Ceased WO2012145382A2 (fr)

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US14/114,811 US20140234860A1 (en) 2011-04-20 2012-04-18 Measurement of pka for cancer characterization

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US201161477593P 2011-04-20 2011-04-20
US61/477,593 2011-04-20

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WO2012145382A2 true WO2012145382A2 (fr) 2012-10-26
WO2012145382A3 WO2012145382A3 (fr) 2013-02-07

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WO2012145399A2 (fr) * 2011-04-20 2012-10-26 Traxxsson, Llc Procédés de diagnostic d'un cancer chez un patient

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* Cited by examiner, † Cited by third party
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GB9704444D0 (en) * 1997-03-04 1997-04-23 Isis Innovation Non-invasive prenatal diagnosis
US7135333B1 (en) * 1997-08-07 2006-11-14 Thomas Jefferson University Compositions that specifically bind to colorectal cancer cells and methods of using the same
US6355623B2 (en) * 1998-09-24 2002-03-12 Hopital-Sainte-Justine Method of treating IBD/Crohn's disease and related conditions wherein drug metabolite levels in host blood cells determine subsequent dosage
JP2000325086A (ja) * 1999-05-21 2000-11-28 Igaku Seibutsugaku Kenkyusho:Kk リン酸化酵素活性の測定方法
WO2000079281A1 (fr) * 1999-06-18 2000-12-28 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Proteine kinase ampc dependante extracellulaire utilisee pour le diagnostic, le pronostic et le traitement du cancer
US20090275546A1 (en) * 2008-04-10 2009-11-05 Istituto Superiore Di Sanita Diagnostic tests and personalized treatment regimes for cancer stem cells

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WO2012145382A3 (fr) 2013-02-07

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